Despite their importance as major viral pathogens in both animals and plants and as a potential resource for biological tools, little is known about the functions of genes encoded by positive strand RNA viruses. To address this serious limit to further progress in virology, we have developed a novel in vitro transcription system which efficiently expresses infection from complete cDNA clones of brome mosaic virus (BMV), a well-characterized positive strand RNA plant virus. This system provides the means to apply powerful recombinant DNA techniques to the in vivo molecular genetics of multicomponent RNA viruses, creating unprecedented opportunities for the study of their gene functions. To utilize these opportunities, established techniques of molecular biology and virology will be applied to existing infectious cDNA clones to efficiently generate and isolate conditional mutants distributed over the entire BMV genome. The phenotypic defects of these mutants will be characterized under nonpermissive conditions by assaying viral translation, RNA replication, assembly and transport with defined biochemical procedures. These analyses, in conjunction with complementation tests, will be used to identify all distinct functions encoded by the viral genome. In vivo recombination of restriction fragments between wild type and mutant clones by standard genetic engineering techniques will be used to assign these functions to specific domains of coding or noncoding sequence. Where appropriate, such mapping will be continued to the nucleotide level by sequence comparisons. Preliminary studies show that gene products encoded by BMV share strong amino acid homology with gene sequences from a surprising range of important plant and animal viruses, implying that the homologous proteins have strongly related functions among the different viruses. Studies of BMV gene function will thus have widely applicable results.